A Penicillium freii DNA fragment with similarity to the beta-keto-acyl synthase motif of the P. griseofulvum 6-methylsalicylic acid synthase encoding gene (MSAS) was identified by screening a cosmid library using a part of MSAS as the probe. Two exons of 93 and 849 bp, encoding a predicted polypeptide of 314 amino acids, and a molecular mass of 33.4 kDa, were identified (PfKS). PfKS was transcribed as a 1.6 kbp messenger. A region corresponding to the MSAS gene encoding essential enzyme activities for the assembly of a polyketide chain was absent in PfKS. Gene disruption experiments in P. freii with a truncated version of PfKS did not result in the detection of homologous integration events.